CN116855486A - Streptococcus prophage lyase lys1644 and application thereof - Google Patents

Streptococcus prophage lyase lys1644 and application thereof Download PDF

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CN116855486A
CN116855486A CN202310898216.3A CN202310898216A CN116855486A CN 116855486 A CN116855486 A CN 116855486A CN 202310898216 A CN202310898216 A CN 202310898216A CN 116855486 A CN116855486 A CN 116855486A
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lyase
streptococcus
lys1644
prophage
cleavage
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牛建蕊
张兴林
马俊飞
李兴帅
于鲁敏
赵玉仲
杨祥朋
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Linyi University
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Abstract

The invention discloses a streptococcus prophage lyase lys1644 and application thereof, which belong to the technical field of preventing and treating related diseases of streptococcus, wherein the amino acid sequence of the lyase lys1644 is shown as SEQ ID NO.1, and the encoding gene of the lyase can be prepared into recombinant vectors or engineering bacteria, thus providing a basis for further application of the lyase lys1644; the lyase has antibacterial activity on streptococcus dysgalactiae, staphylococcus aureus or staphylococcus epidermidis, and provides theoretical basis for preventing and treating cow mastitis and other bacterial infections.

Description

Streptococcus prophage lyase lys1644 and application thereof
Technical Field
The invention relates to the technical field of preventing and treating streptococcal related diseases, in particular to a streptococcal prophage lyase lys1644 and application thereof.
Background
Streptococcus (streptococci) bacteria belong to the gram-positive group, and are widely distributed in the nature, in human and animal faeces and in the nasopharynx of healthy people, among which are a wide variety of pathogenic bacteria. Human pathogenic bacteria include Streptococcus pneumoniae, streptococcus pyogenes, streptococcus faecalis, etc., which can cause clinical diseases such as septicemia, arthritis, meningitis, endocarditis, encephalitis, rheumatic fever, scarlet fever, etc.; the streptococcus agalactiae, streptococcus agalactiae and streptococcus uberis infected with cattle can cause the symptoms of cow mastitis and the like; streptococcus suis can cause meningitis, septicemia, pneumonia, etc. in swine humans; streptococcus equi infection can cause horse to develop horse adenomatosis, cause human wound infection, bacteremia and the like. Streptococcal disease causes great harm to human health and causes great loss to livestock breeding. The presence of antibiotics allows for a certain control of streptococcal infection. However, as antibiotics are used in non-standard manner in human medicine and livestock breeding industries, the resistance of the antibiotics is gradually increased, and thus the emerging multi-drug resistant bacteria have no available antibiotic treatment, so that development of a new class of antibacterial drugs or antibacterial agents for preventing and controlling streptococcus infection is urgently needed.
Phage is a virus capable of infecting a prokaryotic microorganism such as bacteria, archaea, etc., which can rapidly lyse bacteria through a "perforin-lyase" binary system, so that phage and its derived lyase can be used as an alternative antimicrobial agent for antibiotics. Phage lysis spectra are generally narrow and limited in their use because bacteria can rapidly evolve resistance to surface receptors. The lyase derived from phage has a relatively wide cleavage spectrum, can be used as a high-efficiency and safe natural antibacterial agent for preventing and controlling drug-resistant pathogenic bacteria.
The phage lyase is used as an antibacterial agent, can specifically and rapidly kill host bacteria, and the peptidoglycan acted on the surface of the bacteria by the lyase has high in-seed conservation because the peptidoglycan is a key component of the cell wall, so the lyase shows broader cleavage activity and also determines that the bacteria are more difficult to generate resistance to the lyase; meanwhile, when the lyase is used for treating animals infected by pathogenic bacteria, the host animals can generate antibodies against the lyase, but the active center of the lyase is not exposed on the surface epitope generally, so the antibodies against the lyase cannot impair the lysis sterilization effect of the lyase; moreover, the combination of the lyase and other antibacterial agents such as antibiotics has a synergistic antibacterial effect, so that the cleavage spectrum can be widened, the antibacterial activity can be effectively exerted, and the probability of generating resistance of bacteria is reduced.
Aiming at various streptococcus infections related to cow mastitis and diseases caused by other streptococcus such as streptococcus suis, streptococcus equi, streptococcus pneumoniae, streptococcus pyogenes and the like, a broad-spectrum effective streptococcus phage lyase is not developed at present, and is applied to prevention and control of streptococcus infection.
Thus, how to provide a streptococcal phage lyase against the spectrum of streptococci is a problem that needs to be addressed by those skilled in the art.
Disclosure of Invention
In view of the above, the invention provides a streptococcus prophage lyase lys1644, which has antibacterial activity on streptococcus dysgalactiae, staphylococcus aureus and staphylococcus epidermidis related to cow mastitis, and provides a theoretical basis for preventing and treating cow mastitis and bacterial infection.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
a streptococcus prophage lyase lys1644, wherein the amino acid sequence of the lyase lys1644 is shown as SEQ ID NO. 1.
Preferably, the domain of the streptococcal prophage lyase lys1644 comprises an N-terminal glucosamidase cleavage domain, an intermediate CHAP cleavage domain and a C-terminal SH3-5 binding domain.
Preferably, the N-terminal Glucosaminidase cleavage domain is amino acids 2 to 145, the middle CHAP cleavage domain is amino acids 163 to 296, and the C-terminal SH3-5 domain is amino acids 322 to 390.
As the same invention conception as the technical scheme, the invention also claims the application of the streptococcus prophage lyase lys1644 in preparing related medicines for cracking streptococcus dysgalactiae, staphylococcus aureus or staphylococcus epidermidis.
As the same inventive concept as the above technical scheme, the invention also claims the application of streptococcus prophage lyase lys1644 in preparing antibacterial agent for treating cow mastitis and other bacterial infections.
As the same invention conception as the technical scheme, the invention also claims the application of the gene encoding the amino acid sequence shown in SEQ ID NO.1 in preparing related medicines for cracking streptococcus dysgalactiae, staphylococcus aureus or staphylococcus epidermidis.
Preferably, the nucleotide sequence of the gene is shown as SEQ ID NO. 2.
As the same invention concept as the technical scheme, the invention also claims a specific primer for amplifying the coding gene of the streptococcus prophage lyase lys1644, wherein lys1644-F and lys1644-R are respectively shown as SEQ ID NO. 3-SEQ ID NO. 4.
A medicament comprising streptococcus prophage lyase lys1644, the amino acid sequence of which lys1644 is shown in SEQ ID No.1, the concentration of use of streptococcus prophage lyase lys1644 being 50 μg/mL.
As the same inventive concept as the technical scheme, the invention also claims the application of streptococcus prophage lyase lys1644 in preparing biological materials for cracking streptococcus dysgalactiae, staphylococcus aureus or staphylococcus epidermidis, wherein the biological materials comprise recombinant expression vectors and recombinant engineering bacteria; the recombinant expression vector is formed by recombining an expression vector and a coding gene shown as SEQ ID NO. 2; the recombinant engineering bacteria are formed by transforming the recombinant expression vector into host escherichia coli.
The invention also provides a preparation method of the streptococcus prophage lyase lys1644, which comprises the following specific steps:
(1) Amplifying the nucleotide sequence of prophage lyase lys1644 by using streptococcus dysgalactiae SD24 genome as a template;
(2) Cloning the nucleotide sequence obtained in the step (1) into an expression vector to obtain a recombinant expression vector;
(3) Transforming the recombinant expression vector obtained in the step (2) into competent cells of escherichia coli to obtain recombinant engineering bacteria;
(4) Inducing and expressing lysase lys1644 by using recombinant engineering bacteria;
(5) Extracting and purifying the lyase lys1644 obtained in the step (4).
Preferably, the cleavage method used in the cloning of the cleavage enzyme lys1644 nucleotide sequence is BamHI and KpnI double cleavage.
Preferably, the expression vector is pEC, C-terminally with a 6xHis tag; with T7 lac promoter, ni was used + And (5) purifying by affinity chromatography. The vector may be any other vector suitable for expressing a foreign gene in E.coli, such as pET series, pGEX series, pMAL series or pBAD series.
Preferably, the engineering bacteria are escherichia coli BL21 (DE 3) or escherichia coli Rosseta and other expression host bacteria.
Preferably, the inducer used for inducing expression is IPTG, the induction concentration is 0.5mmol/L, and the induction time is 16 h.
Preferably, the culture temperature of the engineering bacteria is 37 ℃ and the induction expression temperature is 25 ℃.
Compared with the prior art, the pre-phage lyase lys1644 is cloned from the streptococcus dysgalactiae SD24 genome, and the soluble lyase is induced and expressed by adopting a prokaryotic expression mode. The lyase lys1644 has broad-spectrum antibacterial effect on streptococcus dysgalactiae, staphylococcus aureus or staphylococcus epidermidis, and lays a foundation for preventing and treating the bacterial infection diseases. The invention can obtain a large number of products with definite enzyme activity and physicochemical properties, has low production cost and strong product activity, and can meet the production requirements of popularization and application.
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In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required to be used in the embodiments or the description of the prior art will be briefly described below, and it is obvious that the drawings in the following description are only embodiments of the present invention, and that other drawings can be obtained according to the provided drawings without inventive effort for a person skilled in the art.
FIG. 1 is a diagram showing SDS-PAGE results for prokaryotic expression and purification of the lyase protein lys1644; wherein M is a standard protein molecular weight marker; 1 is engineering strain whole protein of a transformation empty vector; 2 is the recombinant engineering strain total protein of the transformed recombinant expression plasmid; 3,4 is the result of purification of the lyase protein lys1644;
FIG. 2 is a schematic diagram showing the sequence structure of lys1644 protein; wherein, glucosaminidase is the 1 st cleavage domain, CHAP is the second cleavage domain, SH3-5 is the binding domain;
FIG. 3 is a diagram showing the perspective structure of the alpha fold predicted Streptococcus prophage lyase lys 644;
FIG. 4 is a graph showing the in vitro bacteriostatic effect of lysase lys1644 at various concentrations on Streptococcus dysgalactiae;
FIG. 5 is a graph showing the results of pH stability detection of lysase lys1644;
FIG. 6 is a graph showing the results of temperature stability of lysase lys1644.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1 genomic sequence amplification and recovery
Whole genome sequencing was performed on streptococcus agalactiae SD24 isolated from dairy cows with mastitis milk, the prophage genome was located in the genome by a belief analysis, and the full-length nucleotide sequence of lyase lys1644 was retrieved from the prophage.
The nucleotide sequence shown as Seq ID No.2 was amplified using the primer pair lys 1644-F/R.
The upstream primer lys1644-F:5 ， -aggagatataccatgggatccatgacctttttagataacat-3 ， As shown in SEQ ID NO. 3.
The downstream primer lys1644-R:5 ， -aaaatacaggttttcggtaccatttaatttaccccaaagac-3 ， As shown in SEQ ID No. 4.
The primer sequence contains homologous arm fragments at the connecting sites of the expression vector, and the underlined parts are the homologous fragments.
The reaction system is shown in Table 1.
TABLE 1 PCR amplification System
Reaction conditions:
after the amplification is completed, the PCR amplification product is detected by 1% agarose gel electrophoresis, and the target fragment amplified by PCR is recovered by using a Noruzan rubber cutting recovery kit.
EXAMPLE 2 ligation and transformation of recombinant expression plasmids competent cells
1) The expression vector was subjected to double cleavage, and the cleavage system is shown in Table 2.
TABLE 2 enzyme digestion system
The enzyme digestion process is as follows: and (3) carrying out water bath for 4 hours at 37 ℃, verifying the enzyme digestion system by 1% agarose gel electrophoresis, and recovering the gel to obtain the carrier with the sticky end.
Based on the target gene PCR gel recovery product and plasmid concentration after cleavage, 20. Mu.L of one-step cloning ligation system was determined as shown in Table 3.
Table 3 connection system
2) The ligation systems in Table 3 were mixed and ligated for 30min at 37 ℃. mu.L of the ligation product was added to 100. Mu.L of E.coli DH 5. Alpha. Competent cells, gently mixed and left on ice for 30min. Then rapidly placed on ice for 2min after heat shock at 42 ℃ for 90 s. 890. Mu.L of LB medium was added thereto, and the culture was continued at 37℃for 1 hour with shaking. After centrifugation at 5000rpm for 5min, 900. Mu.L of the supernatant was discarded, and the remaining 100. Mu.L of the resuspended broth was used and spread evenly on a solid LB medium containing Kan (final concentration 50. Mu.g/mL) and incubated in an incubator at 37℃overnight with inversion.
3) And (3) selecting a single colony on a Kan plate, inoculating the single colony into an LB liquid culture medium containing Kan for culture, identifying positive bacterial liquid through PCR after bacterial suspension is obtained, simultaneously sending a PCR identification system to Shanghai biological stock company for sequencing, and comparing a sequencing result with a target gene sequence. After overnight incubation of the properly sequenced recombinant DH 5. Alpha. Strain, the recombinant plasmid was extracted using the Norfluzamide plasmid extraction kit.
4) 10. Mu.L of the recombinant plasmid was added to 100. Mu.L of competent cells of E.coli BL21, and the mixture was left on ice for 30min after mixing. Then rapidly placed on ice for 2min after heat shock at 42 ℃ for 90 s. 890. Mu.L of LB medium was added thereto, and the culture was continued at 37℃for 1 hour with shaking. 100. Mu.L of the bacterial liquid was uniformly spread on a solid LB medium containing Kan (final concentration: 50. Mu.g/mL), and cultured in an incubator at 37℃overnight with inversion.
EXAMPLE 3 prokaryotic expression and purification of the lyase protein lys1644
And (3) picking a single colony on a Kan plate, inoculating the single colony into an LB liquid culture medium containing Kan for culture, and identifying positive bacterial liquid through PCR after bacterial suspension is obtained. Transferring positive bacterial liquid into LB liquid culture medium, shake culturing at 37deg.C to OD 600 After about 0.6-0.8, IPTG was added at a final concentration of 0.5mmol/L and expression was induced by shaking overnight at 25 ℃. An expression engineering bacterium transformed into an empty plasmid was used as a negative control. After the recombinant engineering bacteria are subjected to expansion culture and induced expression, the bacteria are cracked by ultrasonic crushing. After centrifugation at 12000rpm at 4℃for 10min, the supernatant was removed and analyzed by SDS-PAGE. As a result, as shown in FIG. 1, the recombinant engineering bacteria showed thicker bands at 45kDa and 30kDa (lane 2) compared with the empty plasmid engineering bacteria (lane 1).
The cells were resuspended in 50mM imidazole wash, sonicated and lysed, and the supernatant was taken using Ni + The affinity chromatography column is used for purifying the lyase protein. Balancing the chromatographic column with balancing solution for 2-3 times, and loading the supernatant onto the column for multiple times to make target protein and affinity chromatography Ni + The filler was fully bound and washed 6 times with 50mM imidazole to wash off the contaminating proteins. The target protein was then eluted using 200mM imidazole and the results of protein purification are shown in lanes 3 and 4 of FIG. 1. The amino acid sequence of the purified lyase protein lys1644 is shown as SEQ ID NO.1, and the coding gene is shown as SEQ ID NO. 2.
MTFLDNIKQGCLDGWAKYKILPSLTAAQAILESGWGKHAPHNALFGIKADSSWAGKSFNTKTQEEYQPGVMTDIVDRFRAYDSWTDSIFDHGKFLNDNPRYQAVVGETDYKKACHAIKDAGYATASGYAELLIQLIEENDLQKWDDEAIGGKEKQMISSQCREVIEFFINLANAGMGVDKDNFAGWQCADVPCYAAKHWFGVDLWGNAIDLLDSAAAAGWEVHRMPTDANPRAGAFFVQSVPYHQFGHTGIVIEDSDGYTMRTIEQNIDGNADALYVGAPARFNTRDFTGVIGWFYPPYQGDAVTQTVSTEPQTSDTIVETPKSGTFTLDVAEINIRRWPSLASEVVGSYKQGDTVGFDSEGYANGYYWISYVGGSGKRNYLAIGQTDKDGNRISLWGKLN, as shown in SEQ ID NO. 1.
atgacctttt tagataacat taagcaaggc tgcttagacg gttgggctaa atacaaaata ttgccatcct tgaccgcagc acaagcaatc ttagagagcg ggtggggtaa acatgcaccg cacaacgctc tgtttggaat taaggcagat agctcttggg caggcaagtc atttaacact aaaactcagg aggagtatca gccaggggtt atgactgaca tagtagatag gtttagagct tatgacagct ggactgacag tatttttgat cacggcaaat ttttaaacga taatccacgg tatcaggctg ttgttggtga gactgactat aaaaaagctt gtcacgctat caaggacgca ggttatgcca cggcaagtgg gtacgcggag ttgcttatcc aactaatcga ggaaaatgac ctacaaaaat gggatgacga agctatcggt ggaaaggaga agcaaatgat tagttctcaa tgtcgagaag ttattgaatt ttttataaat ttggcaaatg ctggtatggg tgttgataaa gataattttg cgggctggca atgtgcagat gtgccttgtt atgcagcaaa gcactggttt ggggtggacc tttggggcaa tgcgattgat ttgctagata gcgctgctgc cgctggttgg gaagtccacc gcatgccgac agatgcaaat ccacgggctg gagcattttt tgtccaatcg gtgccatatc accaatttgg acatacggga attgtcattg aggacagcga cggttatacc atgcgcacga tcgagcaaaa cattgatggc aatgcagatg ccttatatgt aggcgcaccagctcgtttta acactcgtga ttttactggc gttattggtt ggttttaccc accatatcaa ggagatgcag tcacacaaac cgtcagcaca gagccgcaaa cgtctgacac catcgtagag acaccaaaat ctggtacctt tacgctcgat gttgcagaga ttaatattag acgttggcca agcctcgcca gcgaagtagt aggcagctac aagcaaggcg atactgtcgg ctttgacagc gaaggttatg ccaatggcta ctactggatt agctatgtcg gcggctctgg gaaacgcaac tatttagcta ttggtcagac tgataaagac ggaaatcgta tcagtctttg gggtaaatta aat, as shown in SEQ ID NO. 2.
Example 4 cleavage enzyme lys1644 Structure prediction
The amino acid sequence of lysase lys1644 (SEQ ID NO. 1) was submitted to the Pfam online server (https:// Pfam. Xfam. Org /) for prediction of its domain, as shown in FIG. 2; as can be seen from FIG. 2, glucosaminidase is the 1 st cleavage domain, CHAP is the second cleavage domain and SH3-5 is the binding domain; glucosaminidase starts at value 2 and ends at value 145 in the protein sequence; CHAP starts at value 163 and ends at value 296 in the protein sequence; SH3-5 begins at value 322 and ends at value 390 in the protein sequence.
Three-dimensional structure prediction of the pre-streptococcal phage lyase lys1644 was performed using the AlphaFold Colab website (https:// Colab. Research. Google. Com/gitub/sokrypton/ColabFold/blob/main/AlphaFold 2.Ipynb #scrollto=azikiidicacan), submitting its amino acid sequence (SEQ ID No. 1), and mapping its domain using pymol software after downloading the prediction results, as shown in fig. 3.
EXAMPLE 5 analysis of physicochemical Properties of lysase lys1644
The number of amino acids is 401, the relative molecular mass is 44336.26, the theoretical pI is 4.82, and the types, the numbers and the proportion of the amino acids are respectively as follows: ala (A) 40.0%, arg (R) 13.2%, asn (N) 19.7%, asp (D) 33.2%, cys (C) 5.2%, gln (Q) 18.5%, glu (E) 19.7%, gly (G) 40.0%, his (H) 8.0%, ile (I) 25.2%, leu (L) 21.2%, lys (K) 21.2%, met (M) 6.5%, phe (F) 184.5%, pro (P) 143.5%, ser (S) 23.7%, thr (T) 24.0%, trp (W) 13.2%, tyr (Y) 19.4.7%, val (V) 22.5%, pyl (O) 0.0%, sec (U) 0.0%, ile (B) 0.0%, (Z) 0.0%, (X) 0.0%) (X) 0.0.0%) (total number of positively charged residues (Arg+s) 34.0%) +total number of residues; the atomic number composition C is 1987; h is 2969; n is 527; o is 609; s is 11, molecular formula C 1987 H 2969 N 527 O 609 S 11 The total atomic number is 6103, the extinction coefficient is 100060 and the extinction coefficient is M -1 cm -1 In units, measured in 280nm water; the absorbance values were: 0.1% (=1 g/l) 2.257; the N end of the protein sequence is methionine (Met); the estimated half-life is: 30 Hours (mammalian reticulocytes, in vitro).>20 hours (yeast, in vivo).>10 hours (Escherichia coli, in vivo); instability index the Instability Index (II) was calculated to be 38.01, indicating that the protein classification was stable; the Aliphatic index (Aliphatic index) is 70.62 and the overall average hydrophilicity (GRAVY) is-0.390.
Example 6 in vitro bacteriostatic Activity assay and bacteriostatic Spectrometry of lyase lys1644
Streptococcus were cultured in BHI medium to logarithmic phase, the bacterial liquid was centrifuged, and the pellet was washed 2 times with Tris-HCI (pH 7.5), and then the bacterial cells were resuspended with Tris-HCI and OD was adjusted 600 About 0.6, and then concentrated to 1/2 of the original volume. mu.L of the concentrated bacterial solution was mixed with 100. Mu.L of lysase lys1644 at various concentrations (0, 50, 100, 150, 200. Mu.g/mL) in a 96-well plate to adjust the OD of the mixture 600 The value is about 0.6, and the final concentration of the lyase is 0, 25, 50, 75 and 100 mug/mL respectively. Mixing 100. Mu.L of concentrated bacterial liquid and 100. Mu.L of Tris-HClMixing the solutions as control, placing 3 groups of the solutions in parallel in a multifunctional enzyme labeling instrument, standing at 37deg.C for culturing, and measuring OD every 10min 600 Numerical value, OD is plotted 600 A time-dependent curve.
As shown in FIG. 4, OD of the groups treated with lysase lys1644 at different concentrations 600 The value gradually decreases, the bacterial liquid gradually becomes clear, and the OD of the lyase is not added 600 The numerical values are not changed basically. Wherein, the descending trend of the 25 mug/mL lyase group is slightly worse; OD when the concentration of the lyase is more than 50. Mu.g/mL 600 The decrease of the values tends to be consistent, which indicates that the application concentration of 50 mug/mL can achieve the best antibacterial effect.
The commercially available Streptococcus dysgalactiae, staphylococcus aureus and Staphylococcus epidermidis were used for the determination of the cleavage spectrum of the lyase lys1644, respectively. Culturing the strain to be tested at 37 ℃ to logarithmic growth medium phase, centrifuging the bacterial liquid, washing precipitate with Tris-HCI (pH 7.5) for 2 times, and then re-suspending the bacterial cells with Tris-HCI and adjusting OD 600 About 0.6, and then concentrated to 1/2 of the original volume. mu.L of the concentrated bacterial solution was mixed with 100. Mu.L of lysase lys1644 at 100. Mu.g/mL in a 96-well plate to obtain a mixed solution OD 600 The value is about 0.6, the final concentration of the lyase is 50 mug/mL, 100 mu L of concentrated bacterial liquid and 100 mu L of Tris-HCl mixed liquid are used as a control, 3 groups of the concentrated bacterial liquid and the 100 mu L of Tris-HCl mixed liquid are parallel, and the mixture is placed in a multifunctional enzyme-labeled instrument for static culture at 37 ℃. OD was measured at 0h and 1h 600 Numerical value, calculate OD 600 Ratio of decrease in value, in OD 600 > 20% indicates the ability to cleave to determine the cleavage spectrum of the cleavage enzyme lys1644.
The results of the cleavage spectrum are shown in Table 4, and the cleavage enzyme lys1644 has a strong cleavage effect on 3 streptococcus dysgalactiae strains, and in addition, the cleavage enzyme also has a certain cleavage effect on staphylococcus aureus and staphylococcus epidermidis, and the results show that the cleavage enzyme lys1644 has a specific antibacterial capability on streptococcus dysgalactiae in streptococcus.
TABLE 4 cleavage spectrum cleavage results by the cleavage enzyme lys1644
Example 7 identification of the pH and temperature stability of the lyase lys1644
Analysis of the pH stability of the lyase lys 1644: streptococcus were cultured in BHI medium to logarithmic phase, the bacterial solutions were equally divided into 8 groups, and centrifuged, and then buffered with different pH buffers (50 mM NaCl, 50mM Na) 2 HPO 4 The pH was adjusted to 4,5,6,7,8,9, 10, 11) and the pellet was washed 2 times, then resuspended in the corresponding pH buffer and OD was adjusted 600 About 0.6, and then concentrated to 1/2 of the original volume. 100. Mu.L of concentrated bacterial solutions under different pH conditions were mixed with 100. Mu.L of lyase 1ys1644 (prepared by diluting the lyase with Tris-HCI having different pH) at the same pH in a 96-well plate to obtain a mixed solution OD 600 The value is about 0.6, and the final concentration of the lyase is 50 mug/mL. Mixing and rapidly placing in an enzyme-labeled instrument to determine initial A 630 The value is then put in the mixed solution of bacteria and lyase under different pH conditions for incubation at 37 ℃, and the final A is measured after 1h 630 Numerical value, and calculate ΔA 630 Numerical values. As a control, 100. Mu.L of concentrated bacteria and 100. Mu.L of Tris-HCl mixture were used, 3 of which were in parallel.
As a result of measurement, as shown in FIG. 5, the cleavage enzyme lys1644 was used as DeltaA at pH 6 630 The value is highest, the highest cleavage activity is achieved, and the cleavage enzyme activity decreases to different degrees with increasing or decreasing pH value.
Temperature stability analysis of lyase lys 1644: streptococcus were cultured in BHI medium to logarithmic phase, the bacterial liquid was centrifuged, and the pellet was washed 2 times with Tris-HCI (pH 7.5), and then the bacterial cells were resuspended with Tris-HCI and OD was adjusted 600 About 0.6, concentrating to 1/2 of the original volume, standing the concentrated bacterial liquid at 4deg.C, 25deg.C, 37deg.C, 42deg.C and 56deg.C for 30min, and simultaneously performing the same treatment on lysase lys1644 to be added at the corresponding temperature. mu.L of the concentrated bacterial solution was mixed with 100. Mu.L of lyase 1ys1644 at 100. Mu.g/mL in a 96-well plate to adjust the OD of the mixture 600 The value is about 0.6, and the final concentration of the lyase is 50 mug/mL. Mixing and rapidly placing in an enzyme-labeled instrument to determine initial A 630 Numerical value, then the mixed solution of bacteria and lyase is placed at different temperaturesIncubation at a temperature of 1h followed by determination of A 630 Final value, and calculate ΔA 630 Numerical values. As a control, 100. Mu.L of concentrated bacteria and 100. Mu.L of Tris-HCl mixture were used, 3 of which were in parallel.
As a result of measurement, as shown in FIG. 6, the cleavage enzyme lys1644 was DeltaA at 37 ℃ 630 The value is highest, the highest cleavage activity is achieved, and the cleavage enzyme activity decreases to different degrees with increasing or decreasing temperature. Under all the measured temperature conditions, the lyase lys1644 has higher cleavage activity and better temperature stability.
In summary, the optimal enzymatic activity condition for the lyase lys1644 was ph=6, 37 ℃. And the lyase lys1644 has better temperature stability.
In the present specification, each embodiment is described in a progressive manner, and each embodiment is mainly described in a different point from other embodiments, and identical and similar parts between the embodiments are all enough to refer to each other.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims (7)

1. The streptococcus prophage lyase lys1644 is characterized in that the amino acid sequence of the lyase lys1644 is shown as SEQ ID NO. 1.
2. A streptococcus prophage lyase lysates 1644 according to claim 1, wherein the domain of streptococcus prophage lyase lysates 1644 comprises an N-terminal glucosamidase cleavage domain, an intermediate CHAP cleavage domain and a C-terminal SH3-5 binding domain.
3. A streptococcus prophage lyase lysase 1644 according to claim 2, wherein the N-terminal glucosamidase cleavage domain is amino acids 2 to 145, the middle CHAP cleavage domain is amino acids 163 to 296 and the C-terminal SH3-5 domain is amino acids 322 to 390.
4. Use of the streptococcus prophage lyase lys1644 according to any of claims 1-3 for the preparation of a related medicament for the lysis of streptococcus dysgalactiae, staphylococcus aureus or staphylococcus epidermidis.
5. Use of the streptococcus prophage lyase lys1644 according to any of claims 1-3 for the preparation of an antibacterial agent for the treatment of bovine mastitis.
6. A medicine is characterized by comprising streptococcus prophage lyase lys1644, wherein the amino acid sequence of the lyase lys1644 is shown as SEQ ID NO.1, and the use concentration of the streptococcus prophage lyase lys1644 is 50 mug/mL.
7. Use of the streptococcus prophage lyase lys1644 according to any of claims 1-3 for the preparation of a biomaterial for the lysis of streptococcus dysgalactiae, staphylococcus aureus or staphylococcus epidermidis, said biomaterial comprising a recombinant expression vector and a recombinant engineering bacterium; the recombinant expression vector is formed by recombining an expression vector and SEQ ID NO.2 sequence; the recombinant engineering bacteria are formed by transforming the recombinant expression vector into host escherichia coli.
CN202310898216.3A 2023-07-21 2023-07-21 Streptococcus prophage lyase lys1644 and application thereof Pending CN116855486A (en)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101275146A (en) * 2008-05-07 2008-10-01 浙江树人大学 Production method for streptococcus specific bacteriophage lyase
CN102512670A (en) * 2003-03-04 2012-06-27 英特塞尔股份公司 Streptococcus pyogenes antigens
US20220347241A1 (en) * 2021-04-30 2022-11-03 Shihezi University Preparation of phage cocktail as therapeutic agent for cow mastitis and use thereof
CN116355887A (en) * 2023-05-16 2023-06-30 临沂大学 Streptococcus prophage lyase lys733 and application thereof

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102512670A (en) * 2003-03-04 2012-06-27 英特塞尔股份公司 Streptococcus pyogenes antigens
CN101275146A (en) * 2008-05-07 2008-10-01 浙江树人大学 Production method for streptococcus specific bacteriophage lyase
US20220347241A1 (en) * 2021-04-30 2022-11-03 Shihezi University Preparation of phage cocktail as therapeutic agent for cow mastitis and use thereof
CN116355887A (en) * 2023-05-16 2023-06-30 临沂大学 Streptococcus prophage lyase lys733 and application thereof

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
SINGH 等: "The Prophage-encoded Hyaluronate Lyase Has Broad Substrate Specificity and Is Regulated by the N-terminal Domain", THE JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 289, no. 51, 19 December 2014 (2014-12-19), pages 35225 - 35236 *
XU 等: "Identification and Characterization of a Novel Prophage Lysin against Streptococcus dysgalactiae", MOLECULES, vol. 29, no. 3411, 20 July 2024 (2024-07-20), pages 1 - 10 *
朱丹 等: "噬菌体裂解酶作用机制及用于细菌感染治疗的研究进展", 基础医学与临床, vol. 38, no. 2, 29 January 2018 (2018-01-29), pages 241 - 245 *
涂传灯 等: "鱼源副乳房链球菌前噬菌体裂解酶Sply828 的抗菌活性", 微生物学通报, vol. 49, no. 5, 28 January 2022 (2022-01-28), pages 1719 - 1730 *

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